Serum calprotectin as a marker of neonatal sepsis: a hospital-based cross-sectional diagnostic study

Background Despite significant advances in neonatal care, neonatal sepsis remains a major contributor to mortality, morbidity, and protracted hospitalization. The development of early possible diagnostic indicators for newborn sepsis is critical. Since calprotectin participates in major biological processes, it could be a diagnostic marker for infection/inflammation. This study aimed to estimate serum calprotectin in neonates with clinical sepsis. In addition, we compared serum calprotectin with standard sepsis markers and serum procalcitonin to evaluate its diagnostic accuracy. Methods A hospital-based cross-sectional diagnostic study of neonates identified with clinical sepsis using standard criteria was carried out. We compared estimated serum calprotectin levels to serum procalcitonin levels and conventional sepsis markers (leucocyte count, blood culture, immature to total neutrophil ratio, and C- reactive protein). We used SPSS version 25 to analyze the data. To examine diagnostic accuracy and determine a cut-off value for serum calprotectin, we used the receiver operating characteristics (ROC) curve. Results Of the 83 subjects included, 36.5% (30/83) had blood culture positive status, the median value of serum calprotectin being 0.93 ng/ml (0.67 to 1.3). Respiratory, cardiovascular, and gastrointestinal instabilities were present in 67.5% (56/83), 59% (49/83), and 50.1% (42/83) cases, respectively. The median values of serum calprotectin, procalcitonin, TLC, and I/T ratio between neonates withpositive blood culturesand negative culturesdid not differ significantly.. On ROC, calprotectin was not predictive for blood culture positivity (sensitivity: 50%; specificity: 44% at 0.83 ng/ml of serum calprotectin) and C-reactive protein (CRP) levels (sensitivity: 57%; specificity: 67% at serum calprotectin levels of 0.89 ng/ml). However, compared with serum procalcitonin, serum calprotectin at 1.2 ng/ml had sensitivity and specificity of 60% and 73%, respectively. Conclusions Serum calprotectin did not show a distinct advantage over the existing sepsis markers. Serum calprotectin level at 1.2 ng/ml had a sensitivity and specificity of 60% and 73%, respectively, compared to serum procalcitonin in detecting neonatal sepsis.


Introduction
Sepsis is a severe and potentially lethal organ dysfunction often produced by an inadequate host response to an infection. 1eonates are a unique cohort of populations showing differences in physiology and immunology between children and adults.Even though the last decade has shown a substantial reduction in neonatal mortality globally, septicemia continues to be a significant contributor, accounting for 11 million neonatal deaths yearly. 2 In neonates the onset of sepsis is often quiet with minimal unclear and nonspecific signs.An accurate and early diagnosis plays a critical role.It is for this purpose that a host of novel biomarkers are being explored.The most significant aspect is that they are markers of adaptive immunological responses that are not well established during the initial post-natal period.The gold standard for organism isolation remains to be blood culture.Nevertheless, the culture results are only available after 48 hours and, sometimes, fail to show the growth of microbes despite the clear clinical picture of sepsis.Hence, neonatal sepsis remains challenging for clinicians to ensure accurate diagnosis at the appropriate time.
Calprotectin, also known as MRP 8/14, S100A8/S100A9, is a zinc and calcium-binding protein heterodimer.It is primarily located in the cytosolic neutrophil fraction and comprises nearly 30-40% of the protein content. 3It is released into the circulation due to exocytosis of granules from activated neutrophils. 4Calprotectin intracellular roles include activation of neutrophilic NADPH oxidase and cytoskeletal regulation of phagocyte migration. 5,6It contains apoptosisinducing, antibacterial, proinflammatory, and oxidant-scavenging properties. 7,8In vitro studies have demonstrated bacteriostatic, fungi-static, and resistance to enzymatic degradation.[11][12] Calprotectin is secreted into circulation by innate immune system cells immediately following a host-pathogen interaction.An enzyme-linked immunosorbent assay (ELISA) test can detect it.Its potential use in the diagnosis of various inflammatory diseases is being investigated.4][15][16] The literature on its diagnostic value in newborn sepsis in the Indian context is, however, scarce.
Procalcitonin is one of the most widely studied markers in sepsis.Monocytes and hepatocytes produce procalcitonin, which rises within four hours and has a half-life between 25 and 30 hours. 17It is thus a reliable indicator of sepsis compared to conventional markers.
According to a comprehensive review and meta-analysis by Vouloumanou et al., procalcitonin has a pooled sensitivity and specificity of 81% and 79% in identifying newborn sepsis. 18According to a recent meta-analysis by Ruan et al., procalcitonin paired with C-reactive protein (CRP) or presepsin was more accurate in diagnosing newborn sepsis.They also stated the various cut-off levels used to define neonatal sepsis ranged from 0.5 to 2.2 ng/ml. 19Procalcitonin levels increase physiologically in the first 24 hours of life, with elevations additionally seen in non-infectious causes such as trauma, surgery, and respiratory distress syndrome.These confounding factors limit the utility of procalcitonin in neonatal sepsis, specifically in early-onset sepsis. 15is study aimed to estimate calprotectin levels in newborns with clinical sepsis and compare its diagnostic accuracy with other sepsis markers such as blood culture, CRP, and serum procalcitonin.

Study setting
We conducted cross-sectional diagnostic research at Tertiary Neonatal Critical Care Units linked to Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India.We included admitted neonates with clinical sepsis diagnosed by clinical criteria 20 between December 2018 to September 2020 by convenient sampling.

Study design
This research follows the Standards for Reporting Diagnostic Accuracy (STARD) 21 statement guidelines.The reporting guidelines contain a completed STARD checklist. 22Figure 1 depicts the study flow according to STARD criteria. 22

REVISED Amendments from Version 1
Revisions were made to the methodology, results, and discussion section based on the comments/suggestions from the reviewers.References were added and updated.
Any further responses from the reviewers can be found at the end of the article

Sample size
Based on a prior study by Terrin et al., 13 where the sensitivity of serum calprotectin levels in predicting neonatal sepsis was 89% and using a normogram with 10% absolute precision, 95% confidence interval, with 10% non-responsive rate, the sample size was estimated to be 77 and rounded off to 80.

Ethics and data collection
The 1964 Declaration of Helsinki and its later amendments, as well as other related ethical principles, were followed in the conduct of the study.The Institutional ethics committee of Kasturba Medical College, Mangalore (IEC KMC MLR 10-18/378, dated 17/10/2018) approved the study, and we obtained appropriate hospital authorities' permits.We approached the parents and guardians of newborns who met the inclusion criteria and informed them in their native tongue about the study's goals.We gave the parents a participant information sheet with answers to the most frequently asked queries (as in Extended data). 22We obtained signed informed consent (as in Extended data) 22 if the parents or guardians were willing for their newborns to participate.We gathered the baseline demographic information and neonatal medical history using a validated semi-structured pretested proforma (as in Extended data). 22clusion and exclusion criteria Admitted neonates with clinical sepsis diagnosed as per the clinical criteria were included.We excluded ventilated neonates with respiratory or circulatory failure, previous exposure to antibiotics, preterm less than 32 weeks, had APGAR scores less than "3" or had conditions such as persistent pulmonary hypertension of newborn, congenital malformations, surgical-related disorders including necrotizing enterocolitis (NEC), and severe intracranial bleeding.

Operational definitions
Clinical sepsis in newborns was defined as the presence of two or more of the following characteristics, namely respiratory instability, cardiovascular instability, gastrointestinal instability, temperature instability, sclerema or petechial rash, and nonspecific features. 20We considered sepsis screen to be positive if two or more of the following observed laboratory parameters, namely CRP >6 mg/dl, total leucocyte count (TLC) >20,000Â10 9 /L or <4,000Â10 9 /L and immature to total neutrophil ratio (I/T ratio) >0.2. 20Neonatal sepsis within 72 hours and greater than 72 hours was defined as early-onset sepsis (EOS) and late-onset sepsis (LOS).The sex of the neonate as male or female was determined by external examination of body characteristics by the investigator.

Sample collection
We collected a venous blood sample to estimate TLC, I/T ratio, CRP, blood culture, serum procalcitonin, and serum calprotectin levels.Beckman Coulter's automated system and Nephelometry calculated the total leucocyte count and CRP, respectively.A peripheral smear examination differentiated the leucocytes, and we determined the I/T ratio.For blood cultures, we collected about 1 mL of venous blood under aseptic conditions.We inoculated blood into blood culture medium bottles (BD BACTEC™ PedsPlus™/F culture vials) before being evaluated for growth at regular intervals.
For serum procalcitonin and serum calprotectin levels estimation, we collected an aliquot of 2 ml venous blood in plain tubes, centrifuged it at 5,000 rotations per minute for 15 minutes, and kept the separated serum at -80°C until further processing.Serum calprotectin levels were estimated using the Human CALP (Calprotectin) ELISA Kit (Catalog no: ELK4602) from ELK Biotechnology ® , China, on LISA plus ELISA reader.The detection range for the calprotectin kit was between 31.25 and 2,000 pg/ml, and its sensitivity was 13.7 pg/ml with high specificity.Serum procalcitonin was estimated using the Human PCT (Procalcitonin) ELISA Kit (Catalog No: E-EL-H1492) from Elabscience ® , China, on LISA plus ELISA reader.The procalcitonin kit exhibited a sensitivity of 18.75 pg/ml, a detection range of 31.25 to 2,000 pg/ml, and a coefficient of variation under 10%.We converted the measured values of serum calprotectin and procalcitonin into ng/ml since the conventional expression of serum procalcitonin is in ng/ml.

Statistical analysis
We used IBM SPSS Statistics (RRID:SCR_016479) for Windows, Version 25.0 (IBM Corp., Armonk, NY) to analyze the data.We expressed patient characteristics in proportions.We calculated the median and interquartile ranges for the following parameters: TLC, CRP, I/T ratio, serum calprotectin levels, and procalcitonin levels.By using Mann-Whitney U test, we compared the values of serum calprotectin and other conventional markers of sepsis among culture-positive and culture-negative sepsis.We performed a receiver operator characteristic (ROC) curve to obtain a cut-off for serum calprotectin levels to detect sepsis in neonates when compared with the conventional sepsis parameters/serum procalcitonin.A cut-off of 0.5 ng/ml of procalcitonin was used for the ROC.
Table 2 compares the median serum calprotectin, procalcitonin levels, and conventional markers of sepsis among the blood culture-positive and culture-negative cases.The differences in the median values of serum calprotectin, procalcitonin, TLC, and I/T ratio between blood culture-positive and culture-negative groups were not statistically significant.
The area under the curve (AUC) was 0.39 (S.E.0.06, p=0.1, CI=0.27 to 0.51) when serum calprotectin was compared to blood culture using a ROC curve (Figure 3).We determined the sensitivity and specificity to be 50% and 44%, respectively, at a cut-off level of 0.83 ng/ml.The serum calprotectin ROC curve exhibited an AUC of 0.536 (S.E.0.08, p=0.69,CI=0.37 to 0.70) compared to CRP (Figure 4).We discovered the sensitivity and specificity to be 57% and 67%, respectively, at a cut-off level of 0.89 ng/ml of calprotectin.
Figure 5 displays the ROC curve contrasting serum calprotectin levels and procalcitonin.The AUC was 0.627 (S.E.0.09, p=0.20,CI=0.45 to 0.80).We determined the sensitivity and specificity to be 60% and 73% at a 1.2 ng/ml cut-off level of serum calprotectin.

Discussion
Calprotectin is an innate immune marker; thus, we investigated its significance as a biological marker for the early diagnosis of newborn septicemia.We conducted the study in a district tertiary maternity hospital that provides medical facilities to in and around the Dakshina Kannada district.Many deliveries occur daily with a high probability of developing drug-resistant strains in the hospital environment.The study population primarily comprised SGA babies (term SGA: 15 and preterm SGA: 21).We found the median serum calprotectin levels in clinically septic neonates to be 0.93 ng/ml (IQR 0.67-1.3).15][16]23 The varied broad range of reported values may be because of the differences in the kit employed for estimation.
We observed that the serum calprotectin levels did not show statistically significant difference between culture-positive and negative groups, calprotectin being marginally higher in blood culture-positive group.3 Contrary to previous research, we were unable to determine the precise reason why our study cohort had lower median serum calprotectin levels. Possile explanations included earlier studies that evaluated serum calprotectin in particular neonatal groups, namely very low birth weight 13 and late-onset sepsis.23 The current study, however, comprised a broad sample of newborns with early and late-onset sepsis and a range of birth weights, offering a unique perspective that calls for further subgroup research.Furthermore, the kits used in various studies were different.
Respiratory instability was the most common manifestation seen in more than two thirds of cases (68%) in our study and was similar to the survey by Attia et al. 15 In the current study, calprotectin levels did not significantly differ between males and females, which is consistent with other research. 15,23ood culture-positive cases contributed to 36.5% of the neonates in our research and are in line with the expected culturepositivity rates of 10 to 30% in neonatal sepsis. 24Previous studies have documented blood culture-positive cases in 19.5% 14 , 16.6% 23 and 75% 15 cases.The most common isolates in the present study were Klebsiella and MRSA and were concurrent with the published literature. 14,15 is indeed surprising to note the type of bacterial isolates without antibiotic exposure.Similar to our study, Terrin et al. 13 documented 52 culture-positive proven sepsis in neonates, with six neonates showing growth of candida.These rare Acinetobacter, Citrobacter, and Candida growths were predominantly present in preterm SGA neonates and a single-term SGA baby.We found risk factors such as preterm SGA babies, birth weight less than 1500 grams, intravascular/umbilical catheterization, and NICU stay greater than seven days were prone to have culture-proven sepsis in our study, similar to previous research by Shete et al., 25 and Arora et al. 26 The literature search does not document the serum calprotectin responses in SGA versus AGA neonates or term versus preterm babies.An earlier article by Bartakova et al., 27 demonstrated the serum concentration of calprotectin to be higher in bacterial sepsis when compared to viral infections; however, limited literature is available on serum calprotectin responses between bacterial versus fungal sepsis.
When compared with CRP, blood culture, and serum procalcitonin levels, the ROC curves generated sensitivity and specificity levels with cut-off values of serum calprotectin to detect neonatal sepsis.In the current study, serum calprotectin had a sensitivity of 60% and specificity of 73% compared to serum procalcitonin in identifying newborn sepsis at a 1.2 ng/ml cut-off.There is limited literature that compares serum calprotectin with serum procalcitonin levels in neonates.
Our study showed poor sensitivity and specificity concerning the gold standard blood culture.][16]23 This study's limitations were the lack of a control group for comparison, the relatively smaller sample size, and the sensitivity and detection range of the ELISA kit used to estimate serum calprotectin.Further multicenter studies involving a larger population of neonates are warranted given broad ranges of mean/median values of serum calprotectin, varied ranges of sensitivity and specificity with different cut-off values, and due to varying usages of kits in various studies.

Conclusions
Serum calprotectin is not superior to existing sepsis markers.Serum calprotectin levels equal to and above 1.2 ng/ml had a sensitivity of 60% and specificity of 73% compared to serum procalcitonin in detecting sepsis in our neonatal population.

Reporting guidelines
Open Scientific Framework: STARD checklist for 'Serum calprotectin as a marker of neonatal sepsisa hospital-based cross-sectional diagnostic study'.https://doi.org/10.17605/OSF.IO/6V84E. 22ta are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).
It is surprising to see that sepsis is predominant in the inborn population compared to the outborn.It would be better if there is more elaboration on the base line characteristics of the population -to categorise the population into preterm-term, EOS-LOS, day of investigation, and clinical background (risk factors for sepsis-like PPROM).It helps to understand the study population better and helps to gauge both internal and external validity.

2.
Does the comparison between neonatal sex and septic markers have a significant rationale?
If not the author may consider not mentioning it.To the best of my knowledge, none of the septic biomarkers have sex-specific significance.

3.
Calprotectin has also been explored as a marker of NEC.It is therefore important mentioning any episode of NEC in the study population (especially in the culture-positive group), as it is a bias.Consider adding a subgroup analysis of the marker in the population with GI symptoms and in those without GI symptoms.

4.
Discussion section: Please avoid repeating the statement about the "lower serum calprotectin levels noticed in the study population and possible explanation of heterogenicity" multiple times in the discussion. 1.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Neonatology, Ventilation, POCUS I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
and 2 preterm LGA.Sepsis was early onset (<72 hours) in 36 and late-onset (>72 hours) in 49 babies.The predominant risk factor for sepsis was preterm premature rupture of membranes (PPROM) in 15, prolonged rupture of membranes (PROM) in 6, and maternal urinary tract infections (UTI) in 3 cases.
3. Does the comparison between neonatal sex and septic markers have a significant rationale?If not the author may consider not mentioning it.To the best of my knowledge, none of the septic biomarkers have sex-specific significance.
A). we have made the necessary changes and removed the sentences.
4. Calprotectin has also been explored as a marker of NEC.It is therefore important mentioning any episode of NEC in the study population (especially in the culturepositive group), as it is a bias.Consider adding a subgroup analysis of the marker in the population with GI symptoms and in those without GI symptoms.A). any episode of NEC in the study population was an exclusion criterion to avoid bias and was included in surgical conditions.Currently, we have specified it as -surgical conditions, including NEC (in the inclusion and exclusion criteria).Subgroup analysis of the presence or absence of GI symptoms in neonates did not show any association with biomarkers.The median (IQR) serum calprotectin levels in neonates with the presence and absence of GI symptoms were 0.9 (0.68-1.24) and 0.95(0.67-1.33),respectively, the difference was not statistically significant (p=0.58).
Discussion section: 1. Please avoid repeating the statement about the "lower serum calprotectin levels noticed in the study population and possible explanation of heterogenicity" multiple times in the discussion.
A). we have made the necessary changes as suggested.The median birth weight of the study population of 83 newborns is 2100g.This suggests that the study population was comprised largely of a small gestation-age newborns.It is surprising to note the type of bacterial isolates (Acinetobacter, Citrobacter) and Candida growth (7 of 30) without exposure to prior antibiotics (exclusion criteria).

Competing Interests: Nil
The authors can add the factors influencing serum calprotectin levels -is the serum calprotectin response the same in small and appropriate for age newborns, bacterial vs fungal sepsis, if literature is available.In the discussion, it is mentioned that the calprotectin response was similar in male and female newborns, but this is not presented in the Results.This may be deleted from the discussion.The authors can add the factors influencing serum calprotectin levels -is the serum calprotectin response the same in small and appropriate for age newborns, bacterial vs fungal sepsis, if literature is available.In the discussion, it is mentioned that the calprotectin response was similar in male and female newborns, but this is not presented in the Results.This may be deleted from the discussion.

Is the work clearly and accurately presented and does
A).The median (IQR) serum calprotectin levels in term and preterm neonates were 1(0.67-1.33)and 0.85(0.67-1.23),respectively, and this was not statistically significant (p=0.49).Similarly, we found no significant difference in the response of serum calprotectin levels between SGA and AGA neonates and with bacterial versus fungal sepsis (has been included in the results section) The literature search does not document the serum calprotectin responses in SGA versus AGA neonates or term versus preterm babies.An earlier article by Bartakova et al., demonstrated the serum concentration of calprotectin to be higher in bacterial sepsis when compared to viral infections; however, limited literature is available on serum calprotectin responses between bacterial versus fungal sepsis (has been included in the discussion section) We have deleted the sentence of calprotectin response similar in males and females (discussion section)

Competing Interests: nil
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Figure 2 .
Figure 2. Clinical presentations of neonates with clinical sepsis.

Figure 3 .
Figure 3. Receiver Operator Characteristic (ROC) comparing accuracy of serum calprotectin with blood culture.

Figure 4 .
Figure 4. Receiver Operator Characteristic (ROC) comparing accuracy of serum calprotectin with C-reactive protein.

Reviewer Report 26
June 2023 https://doi.org/10.5256/f1000research.145000.r178894© 2023 Shenoy R.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Rathika Damodara Shenoy 1 Department of Paediatric Surgery, K.S. Hegde Medical Academy, NITTE (Deemed to be University), Mangalore, Karnataka, India 2 Department of Paediatric Surgery, K.S. Hegde Medical Academy, NITTE (Deemed to be University), Mangalore, Karnataka, IndiaNewborn sepsis often presents with non-specific symptoms and signs.Blood cultures are positive in just about one-third.There is a constant search for a sensitive and specific biomarker, and this study has investigated the suitability of serum calprotectin.The comparison has been made against culture-positive sepsis, elevated CRP and procalcitonin.The methodology is well written.The cut-off of PCT used to determine ROC in the study can be added either under Operational definitions or under Statistical analysis sections.

Table 1 .
Median values of septic screening parameters among neonates with clinical sepsis.Blood culture was positive in 36.1% (30/83) of newborns.Among the 30 culture-positive cases, we found bacterial growth in 76% (23/30) and fungal sepsis (Candida species) in 24% (7/30) of newborns.Among the bacterial sepsis, Klebsiella and Methicillin-Resistant Staphylococcus aureus (MRSA) were isolated in six cases each, followed by Acinetobacter in four patients.Further, we documented Citrobacter and Pseudomonas growth in three cases each and Methicillin-sensitive Staphylococcus aureus in one case.

Table 2 .
Comparative characteristics between culture positive and culture negative sepsis.

it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
No competing interests were disclosed.

median birth weight of the study population of 83 newborns is 2100g. This suggests that the study population was comprised largely of a small gestation-age newborns. It is surprising to note the type of bacterial isolates (Acinetobacter, Citrobacter) and Candida growth (7 of 30) without exposure to prior antibiotics (exclusion criteria).
We conducted the study in a district tertiary maternity hospital catering to services in and around the Dakshina Kannada district.Many deliveries occur daily with a high probability of developing drug-resistant strains in the hospital environment.The study population primarily comprised SGA babies (term SGA: 15 and preterm SGA: 21).Yes, it is surprising to note the type of bacterial isolates without antibiotic exposure.Similar to our study, Terrin et al. documented 52 culture-positive proven sepsis in neonates, with six neonates showing growth of candida.These rare Acinetobacter, Citrobacter, and Candida growths were predominantly present in preterm SGA neonates and a single-term SGA baby (included in the discussion section).We found risk factors such as preterm SGA babies, birth weight less than 1500 grams, intravascular/umbilical catheterization, and NICU stay greater than seven days were prone to have culture-proven sepsis in our study, similar to previous research byShete etal., and Arora et al.(included in the discussion section) A) Median (IQR) gestational age was 37.4 (34.9-39.14)weeks.There were 47 term babies (term AGA: 32, term SGA: 15) and 36 preterm babies (preterm AGA: 13, preterm SGA: 21, and preterm LGA: 2) (have been included in the results section).